A Micropropagation Protocol for Ecballium elaterium (L.) A. Rich.
E. Attard1 and H. Attard2
1
Institute of Agriculture, University of Malta, Msida, MSD 06, Malta, and 2College of Agriculture,
Luqa Road, Qormi, QRM08, Malta
Introduction: Squirting cucumber, Ecballium
elaterium (L.) A. Rich. (Cucurbitaceae), is a wild
medicinal plant found abundantly in the
Mediterranean region. It has been utilized as a
rootstock for many cucurbitaceous crops, mainly
attributed to its resistance to abiotic as well as biotic
stress (2). Important pharmacological uses (1, 9) are
attributed to the bitter principles, cucurbitacins (5),
which make the crop inedible. Micropropagation
was aimed at determining the regeneration potential
of this resistant rootstock.
Bonferroni post-hoc test for equality of means. Only
p<0.05 were considered statistically significant.
The results were analyzed statistically by the oneway analysis of variance (ANOVA) followed by the
Results and Discussion: Effects of PGRs on
explants. The effects of the different PGRs or
additives on the nodal explants are shown in table 1.
The best responses for shoot multiplication were with
NAA/BAP combination (Figure 1), followed by Ki
(p<0.05, v=10). BAP responded synergistically with
auxins unlike Ki. Nodal explants produced more
than 5 shoots within 1 month especially with the
NAA/BAP combination. In Gomphrena species, the
index was three or more shoots per nodal segment
after 1 month (8). A low auxin (0.1 mg/l NAA) and a
high cytokinin (5 – 10 mg/l BAP) combination were
optimum. For E. elaterium, decreasing the auxin
concentration decreased the bud multiplication effect.
As regards shoot elongation, the best and significant
response was observed with Ki, BAP and GA3 . In
their absence no elongation took place indicating that
the plant in culture does not store or produce any
endogenous cytokinins. Also cucurbitacins have
anti-gibberellic activity (6) hence intrinsic
gibberellins that may be possibly present are
inhibited by these secondary metabolites. When
NAA was completely omitted from the media, shoot
elongation was noted in all treated shoot explants.
Callus production was seen with all PGRs or
additives except for IAA and charcoal. The 2,4-D/Ki
combination showed significant effects on callus
production with no effects on the other parameters.
This goes in accordance with the observations made
by Esaka (3) on Cucurbita pepo explants. Rooting
was a parameter that posed several problems in the
regeneration of Ecballium elaterium plantlets. In
fact, the whole plantlet was not regenerated in tissue
culture. Although IAA induced rooting, the low
response might be due to the fact that IAA produces a
response in the concentration range between 1 and 30
mg/l (4). Nevertheless, if the auxin had a higher
activity, callus induction and proliferation might have
Cucurbit Genetics Cooperative Report 25: 67-70(2002)
67
Materials and Methods: E. elaterium seeds were
obtained from immature fruit collected in the
Southern region of Malta. The fruit were washed
with tap water for 15 min., surface sterilized with 70
% ethanol for 30 sec, soaked in 10 % hypochlorite
solution for 20 min and rinsed in three changes of
sterile distilled water. Seeds were carefully removed
under aseptic conditions, and placed on Murashige
and Skoog (MS) basal medium (7). Two weeks from
germination, node explants were taken for tissue
culture.
The sectioned node explants were inoculated on MS
medium containing different plant growth regulators
(PGRs) or additives (Table 1), and every 4 weeks the
surviving explants were either subcultured on the
same medium or transferred to a different medium, in
cases of impaired growth. The conditions for growth
were 25 ± 1 °C and 3250 ± 250 lx. Bud
multiplication, shoot elongation, root production and
callus induction and proliferation were observed.
The plantlets were transferred to Jiffy® pots (Sigma,
U.S.A.) and closed in a Phytatray® (Sigma, U.S.A.)
to maintain a high percentage of humidity. With the
emergence of roots from the pot, the plantlets were
transferred to larger pots until flowering.
Cucurbit Genetics Cooperative Report 25: 67-70(2002)
68
Table 1. The overall effects of different mediaz on the different parameters studied.
Percentage for each Stimulus (%)
IA/Ki
NA A/BAP
KI
2,4-D/Ki
IBA
NA A/BAP (1/2)y
MS
IAA
BAP
Charcoal
GA3
Multiplication
Elongation
Callus
Rooting
8.80
26.39x
23.09x
0.00
0.00
14.66
0.00
0.00
0.00
16.50x
10.56
16.70x
7.31
19.27x
0.00
0.00
8.56
0.00
0.00
19.27x
9.63
19.27x
5.76
13.89
3.60
14.39x
14.39x
14.39x
14.39x
0.00
14.39x
0.00
2.88
8.73
0.90
6.55
0.00
26.19x
0.00
26.19x
3.49
26.19x
0.00
5.24
z
The media contained MS medium and 1 mg/L of each PGR or additive listed: indole acetic acid
(IAA), kinetin (Ki), naphthalene acetic acid (NAA), benzylamino purine (BAP), 2,4dichlorophenoxyacetic acid (2,4-D) indole butyric acid (IBA) and gibberellic acids (GA3).
y
NA a?BAP (1/2) contains 0.5 mg/L of BNAA and 1 m g/L of BAP.
x
p,0.05 (v=10).
The experiment was repeated three time with 15 replicates.
Table 2. Time (days) for rooting and repotting for the four treatments.
IAA
Rooting in Jiffy® pots
Repotting
GA3
+R.H.z
-R.H.
+R.H.
-R.H.
10
25
23
37
46
58
63
72
z
Rooting hormone powder (1% NAA and thiram, Secto,UK).
The experiment was repeated three times with 10 replicates.
Cucurbit Genetics Cooperative Report 25: 67-70(2002)
69
set in and hence posing a problem to the rooting
process.
Transfer of explants. Based on the above findings,
the shoot explant grown on GA3 and IAA media
were selected for pot trials, with the use of a rooting
hormone (1 % NAA and thiram, Secto, UK). The
best treatment was IAA cultures treated with
rooting hormone (Table 2). For the IAA with
rooting hormone treatment, flowering took place at
approximately day 62 from transfer to Jiffy® pot.
This was eventually followed by fruiting (Figure 2).
In conclusion, an ideal protocol would involve the
germination of seeds on MS medium followed by
the inoculation of node explants on NAA/BAP for
three consecutive subcultures at 4-week intervals,
elongation on GA3 medium for another 4 week and
an auxin (IAA) shock for 1 week, in tissue culture.
Subsequently shoots should be treated with rooting
hormone (NAA), transferred to Jiffy pots and after
3 to 4 weeks the plantlets should be repotted and
acclimatized for another 4 to 5 weeks. Our results
give a clear protocol for the regeneration of the
squirting cucumber for possible use as a resistant
rootstock for the edible cucurbitaceous crops.
Literature Cited
1. Attard, E., A. Scicluna-Spiteri, M. Grixti and A.
Cuschieri. 1996. The cytotoxic activity of
Cucurbitacin E and Busulphan on ovarian and
stomach cancer cells in vitro: A comparative
study. Xjenza 1:29–34.
2. Bar-Nun, N., and A.M. Mayer. 1990.
Cucurbitacins protect cucumber tissue against
infection by Botrytis cinerea. Phytochem.,
29:787–791.
3. Esaka, M. 1993. Cucurbita pepo (Pumpkin): In
vitro production of ascorbate oxidase. In:
Bajaj, Y.P.S., ed. Biotechnology in Agriculture
and Forestry: Medicinal and Aromatic Plants
IV. Volume 21, Berlin: Springer-Verlag. pp.
115 – 131.
4. Finnie, J.F. and J. Van Staden. 1994. Gloriosa
superba L. (Flame Lily). Micropropagation and
in vitro production of colchicines. In: Bajaj,
Y.P.S., ed. Biotechnology in Agriculture and
Forestry: Medicinal and Aromatic Plants VI.
Volume 26, Berlin: Springer-Verlag. pp. 146–
166.
5. Lavie, D. and E. Glotter. 1971. The
Cucurbitanes; a Group of Tetracyclic
Cucurbit Genetics Cooperative Report 25: 67-70(2002)
Triterpenes; Progress in the Chemistry of
Organic Natural Products XXIX; New York;
U.S.A. - English Translation, pp. 352-354.
6. Morgan, E.D. and I.D. Wilson. 1999. Insect
Hormones and Insect Chemical Ecology. In:
Barton, D.; Nakanishi, K., ed. Comprehensive
Natural Products Chemistry, Volume 8, UK:
Elsevier, pp. 320 – 321.
7. Murashige, T. and F. Skoog. 1962. A revised
medium for rapid growth and bioassays with
tobacco tissue cultures. Physiol. Plant. 15:473–
497.
8. Vieira, C.C.J., H. Mercier, E.P. Chu and R.C.L.
Figueiredo-Ribeiro. 1994. Gomphrena species
(globe amaranth): In vitro culture and
production of secondary metabolites. In: Bajaj,
Y.P.S., ed. Biotechnology in Agriculture and
Forestry: Medicinal and Aromatic Plants VII.
Volume 28, Berlin: Springer-Verlag. pp. 257–
270.
9. Yesilada, E., S. Tanaka, E. Sezik and M.
Tabata. 1988. Isolation of an anti-inflammatory
principle from the juice of Ecballium elaterium.
J. Nat. Prod. 51:504–508
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